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Liquid Biopsy

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Liquid biopsy has the potential to provide information about cancers without invasive biopsy, by using circulating biomarkers. These include proteins, RNA and DNA. They can be used in detection, diagnosis, monitoring and detection of recurrence.[1] A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions (primary and metastases) as well as offering the opportunity to systematically track genomic evolution.[2] Biopsies have been used by clinicians to diagnose and manage disease for 1,000 years.[2] In patients with cancer, biopsies allow the histological definition of the disease and, more recently, have revealed details of the genetic profile of the tumour enabling prediction of disease progression and response to therapies.

 

There are many difficulties in obtaining a tissue biopsy-including the discomfort suffered by the patient, inherent clinical risks to the patient, potential surgical complications and economic considerations-meaning that multiple or serial biopsies are often impractical. In addition, some tumours are not accessible for biopsy, the procedure itself might increase the risk of the cancer ‘seeding’ to other sites,[3] and the procedure might not be recommended for patients receiving antiangiogenic treatment.[4] Even in an ideal situation where several metastatic sites can be biopsied simultaneously, the analysis of the samples can delay the initiation of treatment, and might irremediably jeopardise it. These limitations are particular restraints in the setting of acquired resistance to therapy, as the ability of a clinician to detect therapeutic biomarkers at an early stage would allow a potentially successful change in treatment course. In light of these limitations on the use of single biopsies, new ways to observe tumour genetics and tumour dynamics have evolved. In 1948, the publication of a manuscript that described circulating free DNA (cfDNA) and RNA in the blood of humans was, without knowing it, the first step towards the ‘liquid biopsy’.[5]

 

CIRCULATING TUMOR DNA IN BLOOD AS A LIQUID BIOPSY FOR CANCER

 

The existence of cell-free DNA molecules (cfDNA) circulating in human blood was described in 1948 [5], but it was thirty years later when a higher concentration of cfDNA in cancer patients than in individuals without the disease was reported [6]. This circulating tumor DNA (ctDNA) belongs to the pool of the total cfDNA in blood, but it specifically derives from tumors. In individuals without cancer, fragments of DNA are released into the blood because of cell death (apoptosis) [7], but the concentration of cfDNA is low due to clearance of dead cells by phagocytes. However, tumor patients generally have significantly higher levels of cfDNA because of the high turnover of cancer cells, through both apoptotic and necrotic process [8].

 

Interestingly, both the concentration of ctDNA in blood and the fraction of patients with detectable levels of ctDNA correlate with tumor stage: 47% of patients with cancer at stage I had detectable ctDNA; this fraction increased to 55%, 69% and 82% for patients at stage II, III and IV, respectively [9] (Figure 2A). However, the frequency of patients with detectable ctDNA varies across cancer types [9] (Figure 2B).

 

 

ctDNA has been confirmed to contain DNA mutations of both primary and metastatic lesions[10], such as point mutations, copy number variations and insertions/deletions. Nowadays, personalized cancer therapy–based on genetic alterations – relies on the acquisition of tumor tissue via biopsy and posterior targeted genome sequencing, either before therapy initiation or after resistance appearance[11]. Such profiling of genetic alterations in the ctDNA is defined as “liquid biopsy”. Different studies have shown two main applications of ctDNA liquid biopsies: the assessment of specific mutations that can direct patient management (clinically actionable), and the monitoring of the response and resistance to therapy. [12,13]

 

Detection of CtDNA : 

 

Presently, the most commonly used protocols to obtain cfDNA require approximately 1 ml of serum or plasma (3ml of blood) and preparation should not exceed 4–5h following the blood draw. For plasma preparation, blood must be collected in a tube treated with an anticoagulant, preferably EDTA (ethylenediaminetetraacetic acid). Cells are then removed by centrifugation and the supernatant, or plasma, is removed.[14] Serum is collected after the blood is allowed to clot and following centrifugation the supernatant, or serum, is removed.[15] Circulating DNA is then extracted from the plasma or serum using commercially available kits.

 

Exosomes :

 

ctDNA is not the only entity in blood with information about tumor genetic alterations. In fact, the information obtained from the ctDNA can be further complemented through the analysis of mRNA contained within vesicles (exosomes) or sequestered in tumor-educated platelets.[16]

 

Liquid Biopsies in the Clinic :

 

In the past few years there has been considerable focus on the need for ‘biomarkers’. These biomarkers should be surrogate indicators for a future event, such as disease recurrence, disease progression, or death, and should indicate if a specific treatment will reduce that risk.[17]

 

The Future of Liquid Biopsy :

 

Liquid biopsies present a unique opportunity to advance the understanding of metastatic disease development and to further elucidate the signaling pathways involved in cell invasiveness and metastatic competence. Ultimately, these tests could be used in cancer diagnosis and treatment monitoring. Liquid biopsies could revolutionize cancer care, providing clinicians with rapid access to information on a molecular level at diagnosis, and potentially enabling treatment to be more closely tailored to each patient’s disease state. Liquid biopsies are likely to play a major role in the application and monitoring of therapies that are linked to powerful predictive biomarkers of response in metastatic cancer. Thus, the liquid biopsy will allow for a faster, less invasive and more robust test to help oncologists best manage patients with cancer. [18]

 

Risk versus Benefit :

 

Although liquid biopsies hold the promise of overcoming many of the drawbacks associated with tissue biopsies, it is likely that the latter will remain the gold standard for years to come. Until better technologies are available for testing liquid biopsies, tumor tissue will allow for a more thorough analysis, including the identification of more mutations than what is possible with a blood sample. Advantages and disadvantages to liquid biopsy analysis are summarized below. [18]

 

Advantages and Benefits :

 

  •  Non-invasive method for identification of tumor markers, either as an alternative for patients whose tissue is unable to be biopsied or as an adjunct to evaluate drug response.
  • Possibly less costly than tumor biopsy and analysis.
  • Provides an accurate snapshot of the genomic landscape of the tumor, bypassing issues such as intra tumor heterogeneity, as it is speculated that CTCs and cfDNA carry the driver mutations causing metastases.
  • Able to obtain serial samples during treatment to assess for drug resistance and tumor progression. This is not picked up with a tumor biopsy given that tumor biopsies are generally only done prior to treatment, therefore mutations indicating resistance would not be picked up as these generally arise after starting therapy.
  • Unlike tissue biopsy where the tumor DNA is preserved in formalin-fixed paraffin embedded (FFPE) blocks, DNA cross linking does not occur with liquid biopsy; thereby facilitating tumor DNA sequencing.[19]
    Disadvantages and challenges:
  • Potentially miss biomarkers expressed in the tumor
  • Test variability and assay sensitivity and specificity
  • CTCs are rare, fragile and heterogenous
  • Lack of consensus in technical approaches of choice [19]

 

References:-
http://www.pathogenesisjournal.com/
⦁ Crowley, E. et al. Nat. Rev. Clin. Oncol. advance online publication 9 July 2013; doi:10.1038/nrclinonc.2013.110
⦁ Robertson, E. G. & Baxter, G. Tumour seeding following percutaneous needle biopsy: the real story! Clin. Radiol. 66, 1007–1014 (2011).
⦁ Hompes, D. & Ruers, T. Review: incidence and clinical significance of bevacizumab-related non-surgical and surgical serious adverse events in metastatic colorectal cancer. Eur. J. Surg. Oncol. 37, 737–746 (2011).
⦁ Mandel, P. & Metais, P. Les acides nucleiques du plasma sanguin chez l’homme. C. R. Seances Soc. Biol. Fil. 142, 241–243 (1948). https://www.oncodna.com/wp/wpcontent/uploads/2017/10/FA4_liquidbiopsies_20170825_ks_r2_prod.pdf.
⦁ Leon, S. et al. Free DNA in the Serum of Cancer Patients and the Effect of Therapy. Cancer Res 37, 646–650 (1977).
⦁ Suzuki, N., Kamataki, A., Yamaki, J. & Homma, Y. Characterization of circulating DNA in healthy human plasma. Clin. Chim. Acta 387, 55–58 (2008).
⦁ 4. Jahr, S. et al. DNA Fragments in the Blood Plasma of Cancer Patients : Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells. Cancer Res. 61, 1659–1665 (2001).
⦁ Bettegowda, C. et al. Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Sci Transl Med 6, 1–11 (2014).
⦁ Yi, X. et al. The feasibility of using mutation detection in ctDNA to assess tumor dynamics. Int. J. Cancer 140, 2642–2647 (2017).
⦁ Diaz, L. & Bardelli, A. Liquid biopsies: Genotyping circulating tumor DNA. J. Clin. Oncol. 32, 579–586 (2014).
⦁ Bettegowda, C. et al. Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Sci Transl Med 6, 1–11 (2014).
⦁ Krishnamurthy, N., Spencer, E., Torkamani, A. & Nicholson, L. Liquid Biopsies for Cancer: Coming to a Patient near You. J. Clin. Med. 6, 1–11 (2017).
⦁ Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985–990 (2008).
⦁ Wang, J. Y. et al. Molecular detection of APC,K-ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers. World J. Surg. 28, 721–726 (2004).
16- https://www.oncodna.com/wp/wpcontent/uploads/2017/10/FA4_liquidbiopsies_20170825_kr2_prod.pdf.
17-Brooks, J. D. Translational genomics: the challenge of developing cancer biomarkers. Genome Res. 22, 183–187 (2012).
18- http://www.roche-diagnostics.ch/content/dam/corporate/roche—dia_ch/documents/broschueren/molecular_diagnostics/parameter/05-humangenetik-onkologie/egfr/Liquid%20Biopsy%20Backgrounder.pdf
19- https://www.healio.com/hematology-oncology/learn-genomics/introduction-to-liquid-biopsies/what-is-a-liquid-biopsy